Fig 1.
The two groups providing biopsies for analysis are highlighted in the red box.
Table 1.
Subject demographics of the skin biopsy groups (n = 10).
Fig 2.
Skin site assessment post HD-MAP applications in the 10 biopsy-group subjects.
(A) Erythema scores; (B) Oedema scores; (C) Skin Irritation Index (erythema and oedema scores); (D) Pain scores following HD-MAP applications over the course of the study. Only scores of site 2 were included as it was the only HD-MAP application site that was not biopsied. Depicted are mean ± 95% confidence interval.
Fig 3.
Skin site of two representative subjects post HD-MAP application.
Shown are representative volar forearms from active (left) and placebo HD-MAP groups (right). Day 4 images were taken prior to the biopsy.
Table 2.
Summary of HAI assay results as geometric mean titres (GMTs), seroprotection (%), seroconversion (%) and GMT-fold increase.
Fig 4.
H&E-stained skin biopsies from two representative subjects who received active/vaccine-coated HD-MAPs (upper row) or placebo HD-MAPs (lower row). Biopsies were taken aseptically on day 1 (pre) and day 4 (post) HD-MAP application. Following fixation, biopsies were paraffin-embedded, and 5 μm thick sections were subjected to H&E staining. Pre-MAP sections (left column) showed very little cellular infiltration, whereas post-MAP applications (right column) resulted in substantially more cellular influx (dark spots) within the dermis. Epidermal thickening was also noticeable on day 4, along with scab formation in the stratum corneum (SC), and viable epidermis (VE). Blue arrows: micro-scabs post HD-MAP projections penetrating the skin; orange arrows: cell infiltrates. Scale bar: 500 μm.
Fig 5.
Quantification of cellular infiltration into the skin of active and placebo HD-MAP, pre- and post-HD-MAP application.
(A) Haematoxylin-stained nuclei as proxy for cell infiltrates were indirectly quantified by dividing total cell area measurements by average cell size, and then normalised to the dermal area. (B) Total (DAPI stained) cells per group and time-point. (C) Cumulative cell counts of AF-555-positive stained cells per group and timepoint, where cells positive for specific markers were added up per group and time point. A marked increase in cellular infiltrate in active HD-MAP-treated subjects at day 4, compared to day 1, and compared to the placebo group on both days. Statistical significance was determined by two-way ANOVA; * p<0.05, ** p<0.01.
Fig 6.
Increase of haematopoietic cells into the application site of antigen-coated HD-MAPs between day 1 and day 4.
Haematopoietic cells measured by the presence of CD45 cell surface marker in human skin biopsies taken pre- (day 1 blue) and 3 days (day 4 orange) post HD-MAP application as (A) total cell counts or as (B) percentage of CD45+ cells of all single live cells. Analysed by two-way ANOVA, * p<0.05, *** p<0.001, **** p<0.0001.
Fig 7.
Influx of T cells into the application site of antigen-coated HD-MAPs on/by day 4.
(A) CD45+ population sub-gated for CD3/TCRαβ+ T cell populations in human skin biopsies taken pre- (day 1 blue) and 3 days post- (day 4 orange) HD-MAP application. (B) T cells were further assessed by sub-gating for the presence of CD45RO to quantify effector memory T cells (Tems) in peripheral tissue; (C) HLA-DR, a late activation marker for T cells; (D-H) CD4 and CD8 T cell subtype cell surface markers. (I) Immunofluorescence slides are from two representative subjects who received active HD-MAP (left) or placebo HD-MAP (right) from day 1 and day 4. Biopsies were taken aseptically on day 1 and 4 HD-MAP application. Following fixation, paraffin-embedded 5 μm thick sections were subjected to immunofluorescent staining. DAPI (blue), specific cell surface markers (yellow). Tem: effector memory T cells. Statistical significance assessed by two-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 8.
Influx of non-T cells and DCs into the application site of antigen-coated HD-MAPs by day 4.
(A) CD45+ population sub-gated for CD3/TCRαβ- non-T cell populations in human skin biopsies taken pre- (day 1 blue) and 3 days (day 4 orange) post HD-MAP application. (B) Non-T cells were further sub-gated into B cells (CD19+/CD20+). (C) Non-DC myeloid cells (CD11b+CD11b-). (D) DCs (CD11c+) and corresponding subtypes of (E) myeloid (CD11b+) and (F) lymphoid (CD11b-) populations. The presence of HLA-DR was used to distinguish (G) dermal CD11c+CD11b+ and (H) epidermal CD11c+CD11b- DCs. Statistical significance was assessed by two-way ANOVA, * p<0.05, ** p<0.01.
Table 3.
Summary of changes in marker expression between pre (day 1) and post (day 4) MAP application.
Fig 9.
Assessment of cell infiltrates.
Cells that stained AF555-positive for CD3, CD4, CD8, CD11c, CD14, CD19, CD45RO, CD68, HLA-DR, and Ki-67 were quantified and normalised against DAPI stained cells. Statistical significance was assessed by two-way ANOVA matched by subject on log transformed data; placebo day 1 to day 4 ** p = 0.0011, active day 1 to day 4 **** p<0.0001, active versus placebo day 1 to day 4: ** p = 0.0084.